Project Summary/Abstract Mycobacterium tuberculosis causes nine million new active infections globally each year and led to 1.5 million deaths in 2014. Half a million of the global annual M. tuberculosis cases are estimated to be multidrug- resistant (MDR) or extensively drug resistant (XDR), and it is expected that MDR infections will only increase in prevalence. The current second-line agents used to treat MDR- and XDR-M. tuberculosis fail in more than half of cases. Resistance to front-line agents and a lack of effective, non-toxic second-line agents leaves a large unmet need for robust, fast-acting antitubercular drugs. Our laboratory has previously developed salicyl-AMS, a rationally designed intermediate analog of the aryl adenylation enzyme MbtA. Salicyl-AMS has low nanomolar biochemical inhibitory activity, sub-micromolar antimicrobial activity, and in vivo efficacy. These properties make salicyl-AMS attractive as a novel antibiotic. However, salicyl-AMS currently suffers from rapid clearance, low oral bioavailability, and modest mycobacterial wall permeability. The objective of this project is to optimize this lead compound to develop more potent and drug-like inhibitors with improved physicochemical properties. We will synthesize salicyl-AMS analogs with superior physiochemical parameters through the development of more lipophilic analogs, deletion of heteroatoms, and exploration of non-anionic linker analogs. We will then test these analogs in biochemical and cellular assays in collaboration with the laboratories of Dr. Luis Quadri (Brooklyn College) and Dr. William Bishai (John Hopkins University) for binding affinity, in vitro bacterial growth inhibition and sterilizing capability, mammalian cell cytotoxicity, and mycobacterial wall permeability.13,14 Active analogs will be tested for in vitro pharmacology (Sai Life Sciences) and in vivo pharmacokinetics in collaboration with Dr. Elisa de Stanchina (Memorial Sloan Kettering). We will then use salicyl-AMS and optimized analogs to test the hypothesis that bacteria develop resistance to inhibitors of the biosynthesis of essential ?communal goods? at a lower frequency than traditional antibiotics. If successful, the research proposed herein should result in potent, effective, nontoxic lead compounds for further preclinical development of novel antitubercular drugs. These compounds can probe the propensity of bacteria to develop resistance to drugs that inhibit the biosynthesis of essential communal goods, providing evidence regarding the communal goods/decreased resistance hypothesis. The design strategy for the inhibitors proposed herein can be translated to inhibitor design for additional adenylation enzymes, a large and untapped family of potential antibacterial targets.